Nucleic acid prodrugs

ABSTRACT

The invention relates to deoxynucleotide prodrugs and pharmaceutical preparations thereof. The deoxynucleotide prodrugs include, but are not limited to, 
     
       
         
         
             
             
         
       
     
     The invention further relates to methods of treatment of a mitochondrial DNA (mtDNA) depletion syndrome (MDS) by administering a therapeutically-effective amount of a deoxynucleotide prodrug of the present invention.

REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Stage of International PatentApplication No. PCT/US2016/062271, filed Nov. 16, 2016 which claims thebenefit of U.S. Provisional Patent Application No. 62/255,829, filedNov. 16, 2015, the contents of each of which are fully incorporated byreference herein.

BACKGROUND OF THE INVENTION

Mitochondrial DNA (mtDNA) depletion syndrome (MDS) encompasses a groupof genetic disorders characterized by a severe reduction in mtDNAcontent leading to respiratory chain deficiency in affected tissues andorgans. MDS arises due to defects in mtDNA maintenance caused bymutations in nuclear genes that function in either mitochondrialnucleotide synthesis, deoxyribonucleoside triphosphate (dNTP) metabolismor mtDNA replication. There are also some MDSs with unknownpathophysiology.

Some exemplary MDSs are deoxyguanosine kinase (DGUOK) deficiency,thymidine kinase 2 (TK2) deficiency, mitochondrial neurogastrointestinalencephalomyopathy (MNGIE), mitochondrial DNA polymerase (POLG)deficiencies (including Alpers-Huttenlocher syndrome, SANDO syndrome,MIRAS, etc.), MPV17-related hepatocerebral and RRM2B-related myopathies.Of known mutations, there are over ten genes that have been linked toMDS (TK2, DGUOK, POLG, MPV17, RRM2B, SUCLA2, SUCLG1, TYMP, C10orf2, andSAMHD1).

Direct supplementation with nucleosides, deoxyribonucleosidemonophosphates (dNMPs), deoxyribonucleoside diphosphates (dNDPs) ordNTPs has shown the ability to rescue mtDNA depletion in in vitro modelsof MDS and increase overall survival in animal models of MDS in vivo.However, the pharmacological prospects for nucleosides, dNMPs, dNDPs anddNTPs as practical treatments for MDS in humans are low. The negativelycharged phosphates on dNMPs, dNDPs and dNTPs preclude diffusion acrosscellular membranes. Furthermore, intra- and extracellular phosphataseseffectively dephosphorylate dNMPs, dNDPs and dNTPs to the basenucleoside prior to reaching the desired site of action. Although thebase nucleoside can enter the cell via passive and active transportmechanisms, it cannot by itself address the deficiencies of MDS giventhat phosphorylation of a nucleoside to a dNMP is the rate-limiting stepof nucleotide synthesis and, in many cases, MDS patients lack the enzymeresponsible for this transformation. Such considerations require highdoses of nucleosides, dNMPs, dNDPs or dNTPs to potentially achievetherapeutic benefit.

Thus, there is a need for new therapies for MDS, and in particular fortherapies that can effectively provide dNMPs, dNDPs or dNTPs tomitochondria.

SUMMARY OF THE INVENTION

In certain embodiments, the present invention provides compounds havingthe structure of formula (I):

or a pharmaceutically acceptable salt and/or prodrug thereof. In thestructure of formula (I):

R₁ is aryl or heteroaryl;

R₂ and R₂′, each independently, are hydrogen, alkyl or aralkyl;

R₃ is alkyl or aralkyl;

R₄ is hydrogen or alkyl; or

R₂ and R₄ together with the —C—N— moiety that separates them may form aheterocycle; and

NT is adenine, guanine, cytosine, or thymine, or a nucleobase prodrugmoiety such as an adenine, guanine, cytosine, or thymine prodrug moiety.

Exemplary compounds of Formula (I) include the compounds depicted inTable I.

The invention further relates to pharmaceutical compositions of thesubject compounds, as well as methods of using these compounds orcompositions in the treatment of MDSs such as deoxyguanosine kinase(DGUOK) deficiency, thymidine kinase 2 (TK2) deficiency, mitochondrialneurogastrointestinal encephalomyopathy (MNGIE), mitochondrial DNApolymerase (POLG) deficiencies (including Alpers-Huttenlocher syndrome,SANDO syndrome, MIRAS, etc.), MPV17-related hepatocerebral myopathy, orRRM2B-related myopathy; or in treating a mitochondrial DNA depletionsyndrome linked to a mutation in TK2, DGUOK, POLG, MPV17, RRM2B, SUCLA2,SUCLG1, TYMP, C10orf2, or SAMHD1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of a study on the ability of compounds 15 and1017 of the present invention to rescue mtDNA depletion inpatient-derived fibroblasts.

DETAILED DESCRIPTION OF THE INVENTION

In certain embodiments, the present invention provides compounds havingthe structure of formula (I):

and pharmaceutically acceptable salts and/or prodrugs thereof, wherein:R₁ is aryl or heteroaryl; R₂ and R₂′, each independently, are hydrogen,alkyl or aralkyl; R₃ is alkyl or aralkyl; R₄ is hydrogen or alkyl; or R₂and R₄ together with the —C—N— moiety that separates them may form aheterocycle; and NT is a nucleobase such as adenine, guanine, cytosine,or thymine, or a nucleobase prodrug moiety such as an adenine, guanine,cytosine, or thymine prodrug moiety.

In some embodiments of formula (I), NT is a guanine prodrug moiety withthe following structure:

wherein R₅ is alkyl or aralkyl. In some embodiments, NT is a thymineprodrug moiety with the following structure:

wherein R₅ is alkyl or aralkyl. In some preferred embodiments, NT is themoiety with the following structure:

In some embodiments of formula (I), NT is a nucleobase, such as anatural nucleobase. In some such embodiments, NT is adenine. In othersuch embodiments, NT is guanine. In still other such embodiments, NT iscytosine. In yet other such embodiments, NT is thymine.

In some embodiments of formula (I), R₁ is a C₆-C₂₀ aryl or a 5-20 atomheteroaryl, such as phenyl, naphthyl, or 4-fluorophenyl. In somepreferred embodiments, R₁ is naphthyl. In other preferred embodiments,R₁ is phenyl.

In some embodiments of formula (I), R₂ and R₂′, each independently, isselected from hydrogen, C₁-C₆ alkyl, or C₇-C₁₆ aralkyl, or a naturalamino acid side chain. In some embodiments, R₂ is selected from hydrogenor C₁-C₆ alkyl. In some preferred embodiments, R₂ is hydrogen, methyl,isopropyl, or benzyl, most preferably methyl. In other preferredembodiments, R₂ is a natural amino acid side chain. In some preferredembodiments, R₂′ is methyl. In other preferred embodiments, R₂′ is H.

In some embodiments of formula (I), the carbon to which R₂ is attachedis in the S-configuration. In other embodiments, the carbon to which R₂is attached is in the R-configuration. In some embodiments, the carbonto which R₂ is attached is in the D-configuration. In certain preferredembodiments, the carbon to which R₂ is attached is in theL-configuration (i.e., R₂ is disposed in the L-configuration). Accordingto these embodiments, the remainder of the variables in formula (I) maybe selected as described above and below.

In some embodiments of formula (I), R₃ is selected from C₁-C₆ alkyl orC₇-C₁₆ aralkyl, such as C₁-C₆ alkyl or C₇-C₁₁ aralkyl. In some preferredembodiments, R₃ is hydrogen, methyl, isopropyl, neopentyl, or benzyl.

In some embodiments of formula (I), R₄ is selected from hydrogen orC₁-C₆ alkyl, such as hydrogen or C₁-C₃ alkyl, e.g., methyl, ethyl,propyl, or isopropyl. In some preferred embodiments, R₄ is methyl. Inother preferred embodiments, R₄ is hydrogen.

In some embodiments, R₂ and R₄, together with the —C—N— moiety thatseparates them, form a 5-10-atom heterocycle, such as a 5-atomheterocycle. In some preferred embodiments, R₂ and R₄, together with the—C—N— moiety that separates them, form a pyrrolidine ring, e.g., as inproline.

In some embodiments of formula (I), R₅ is selected from C₁-C₆ alkyl orC₇-C₁₆ aralkyl, such as C₁-C₆ alkyl or C₇-C₁₁ aralkyl e.g., methyl,ethyl, isopropyl, or benzyl. In some preferred embodiments, R₅ is ethyl.In some preferred embodiments, R₅ is methyl.

In certain embodiments, the present invention provides compounds havingthe structure of formula (Ia):

and pharmaceutically acceptable salts and/or prodrugs thereof, wherein:R₁ is aryl or heteroaryl; R₂ is hydrogen, alkyl or aralkyl; R₃ is alkylor aralkyl; R₄ is hydrogen or alkyl; and NT is adenine, guanine,cytosine, or thymine.

In further embodiments of formula (Ia), R₁ is phenyl, naphthyl, or4-fluorophenyl; R₂ is methyl and the carbon to which R₂ is attached isin the L-configuration; R₃ is methyl, benzyl, or isopropyl; or R₄ ishydrogen. In further embodiments, R₁ is phenyl, naphthyl, or4-fluorophenyl; R₂ is methyl and the carbon to which R₂ is attached isin the L-configuration; R₃ is methyl, benzyl, or isopropyl; and R₄ ishydrogen. In some preferred embodiments, R₁ is naphthyl. In somepreferred embodiments, R₁ is phenyl.

In certain embodiments, the invention relates to compounds of thestructures depicted in Table 1 and pharmaceutically acceptable salts andprodrugs thereof.

TABLE 1 A

1

2

3

4

5

6

7

8

9 G

10

11

12

13

14

15

16

17

18 T

19

20

21

22

23

24

25

26

27 C

28

29

30

31

32

33

34

35

36

In some preferred embodiments of formula (I), the compound is Compound1017:

or a pharmaceutically acceptable salt thereof.

In some preferred embodiments of formula (I) or formula (Ia), thecompound is Compound 15:

These compounds are prodrugs of dNMPs, and can be used to treat MDSs, orfor any other purpose for which dNMP prodrugs, or dNMPs themselves, areuseful in the treatment of disease.

These compounds are expected to have desirable physicochemicalproperties, given their calculated log P (octanol-water partition), logS (solubility in water), and TPSA (total polar surface area) values allindicate that they will efficiently cross cell membranes and be readilysolvated in biological fluids. Those calculated values are given inTable 2.

TABLE 2 Compound NT R₁ R₂ R₃ R₄ log P log S TPSA 1 A Ph L-Me Me H 0.34−3.73 173 2 A Ph L-Me Bn H 2.12 −5.49 173 3 A Ph L-Me iPr H 1.14 −4.38173 4 A Np L-Me Me H 1.56 −5.60 173 5 A Np L-Me Bn H 3.34 −7.37 173 6 ANp L-Me iPr H 2.36 −6.26 173 7 A 4-FPh L-Me Me H 0.49 −4.02 173 8 A4-FPh L-Me Bn H 2.28 −5.79 173 9 A 4-FPh L-Me iPr H 1.29 −4.67 173 10 GPh L-Me Me H −0.23 −3.43 189 11 G Ph L-Me Bn H 1.55 −5.20 189 12 G PhL-Me iPr H 0.57 −4.08 189 13 G Np L-Me Me H 0.99 −5.31 189 14 G Np L-MeBn H 2.77 −7.07 189 15 G Np L-Me iPr H 1.79 −5.96 189 16 G 4-FPh L-Me MeH −0.08 −3.72 189 17 G 4-FPh L-Me Bn H 1.71 −5.49 189 18 G 4-FPh L-MeiPr H 0.72 −4.38 189 19 T Ph L-Me Me H −0.15 −2.65 153 20 T Ph L-Me Bn H1.64 −4.42 153 21 T Ph L-Me iPr H 0.65 −3.31 153 22 T Np L-Me Me H 1.07−4.53 153 23 T Np L-Me Bn H 2.86 −6.30 153 24 T Np L-Me iPr H 1.87 −5.18153 25 T 4-FPh L-Me Me H 0.00 −2.95 153 26 T 4-FPh L-Me Bn H 1.79 −4.71153 27 T 4-FPh L-Me iPr H 0.81 −3.60 153 28 C Ph L-Me Me H 0.13 −2.83162 29 C Ph L-Me Bn H 1.91 −4.60 162 30 C Ph L-Me iPr H 0.93 −3.49 16231 C Np L-Me Me H 1.35 −4.71 162 32 C Np L-Me Bn H 3.13 −6.48 162 33 CNp L-Me iPr H 2.15 −5.37 162 34 C 4-FPh L-Me Me H 0.28 −3.13 162 35 C4-FPh L-Me Bn H 2.07 −4.90 162 36 C 4-FPh L-Me iPr H 1.08 −3.78 162

In certain embodiments, compounds of the invention may be prodrugs ofthe compounds of Table I, e.g., wherein a hydroxyl in the parentcompound is presented as an ester or a carbonate, or carboxylic acidpresent in the parent compound is presented as an ester. In certain suchembodiments, the prodrug is metabolized to the active parent compound invivo (e.g., the ester is hydrolyzed to the corresponding hydroxyl, orcarboxylic acid).

In certain embodiments, compounds of the invention may be racemic. Incertain embodiments, compounds of the invention may be enriched in oneenantiomer. For example, a compound of the invention may have greaterthan 30% ee, 40% ee, 50% ee, 60% ee, 70% ee, 80% ee, 90% ee, or even 95%or greater ee. In certain embodiments, compounds of the invention mayhave more than one stereocenter. In certain such embodiments, compoundsof the invention may be enriched in one or more diastereomers. Forexample, a compound of the invention may have greater than 30% de, 40%de, 50% de, 60% de, 70% de, 80% de, 90% de, or even 95% or greater de.

In certain embodiments, the present invention relates to methods oftreatment with a compound of formula (I) or (Ia), or a compound selectedfrom Table I, or a pharmaceutically acceptable salt thereof. In certainembodiments, the method comprises administering the compound to apatient in need thereof. In certain embodiments, the therapeuticpreparation may be enriched to provide predominantly one enantiomer of acompound (e.g., of a compound selected from Table I). Anenantiomerically enriched mixture may comprise, for example, at least 60mol percent of one enantiomer, or more preferably at least 75, 90, 95,or even 99 mol percent. In certain embodiments, the compound enriched inone enantiomer is substantially free of the other enantiomer, whereinsubstantially free means that the substance in question makes up lessthan 10%, or less than 5%, or less than 4%, or less than 3%, or lessthan 2%, or less than 1% as compared to the amount of the otherenantiomer, e.g., in the composition or compound mixture. For example,if a composition or compound mixture contains 98 grams of a firstenantiomer and 2 grams of a second enantiomer, it would be said tocontain 98 mol percent of the first enantiomer and only 2% of the secondenantiomer.

In certain embodiments, the therapeutic preparation may be enriched toprovide predominantly one diastereomer of a compound (e.g., of acompound selected from Table 1). A diastereomerically enriched mixturemay comprise, for example, at least 60 mol percent of one diastereomer,or more preferably at least 75, 90, 95, or even 99 mol percent.

In certain embodiments, the present invention relates to methods oftreatment with a compound selected from Table I, or a pharmaceuticallyacceptable salt thereof. In certain embodiments, the therapeuticpreparation may be enriched to provide predominantly one enantiomer of acompound (e.g., of a compound selected from Table 1). Anenantiomerically enriched mixture may comprise, for example, at least 60mol percent of one enantiomer, or more preferably at least 75, 90, 95,or even 99 mol percent. In certain embodiments, the compound enriched inone enantiomer is substantially free of the other enantiomer, whereinsubstantially free means that the substance in question makes up lessthan 10%, or less than 5%, or less than 4%, or less than 3%, or lessthan 2%, or less than 1% as compared to the amount of the otherenantiomer, e.g., in the composition or compound mixture. For example,if a composition or compound mixture contains 98 grams of a firstenantiomer and 2 grams of a second enantiomer, it would be said tocontain 98 mol percent of the first enantiomer and only 2% of the secondenantiomer.

In certain embodiments, the therapeutic preparation may be enriched toprovide predominantly one diastereomer of a compound (e.g., of acompound selected from Table 1). A diastereomerically enriched mixturemay comprise, for example, at least 60 mol percent of one diastereomer,or more preferably at least 75, 90, 95, or even 99 mol percent.

In certain embodiments, the present invention provides a pharmaceuticalpreparation suitable for use in a human patient, comprising any of thecompounds shown above (e.g., a compound of the invention, such as acompound of Formula (I) or (Ia) or a compound selected from Table 1),and one or more pharmaceutically acceptable excipients. In certainembodiments, the pharmaceutical preparations may be for use in treatingor preventing a condition or disease as described herein. In certainembodiments, the pharmaceutical preparations have a low enough pyrogenactivity to be suitable for use in a human patient.

Compounds of any of the above structures may be used in the manufactureof medicaments for the treatment of any diseases or conditions disclosedherein.

Definitions

The term “acyl” is art-recognized and refers to a group represented bythe general formula hydrocarbylC(O)—, preferably alkylC(O)—.

The term “acylamino” is art-recognized and refers to an amino groupsubstituted with an acyl group and may be represented, for example, bythe formula hydrocarbylC(O)NH—.

The term “acyloxy” is art-recognized and refers to a group representedby the general formula hydrocarbylC(O)O—, preferably alkylC(O)O—.

The term “alkoxy” refers to an alkyl group, preferably a lower alkylgroup, having an oxygen attached thereto. Representative alkoxy groupsinclude methoxy, ethoxy, propoxy, tert-butoxy and the like.

The term “alkoxyalkyl” refers to an alkyl group substituted with analkoxy group and may be represented by the general formulaalkyl-O-alkyl.

The term “alkenyl”, as used herein, refers to an aliphatic groupcontaining at least one double bond and is intended to include both“unsubstituted alkenyls” and “substituted alkenyls”, the latter of whichrefers to alkenyl moieties having substituents replacing a hydrogen onone or more carbons of the alkenyl group. Such substituents may occur onone or more carbons that are included or not included in one or moredouble bonds. Moreover, such substituents include all those contemplatedfor alkyl groups, as discussed below, except where stability isprohibitive. For example, substitution of alkenyl groups by one or morealkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups iscontemplated.

An “alkyl” group or “alkane” is a straight chained or branchednon-aromatic hydrocarbon which is completely saturated. Typically, astraight chained or branched alkyl group has from 1 to about 20 carbonatoms, preferably from 1 to about 10 unless otherwise defined. Examplesof straight chained and branched alkyl groups include methyl, ethyl,n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl,pentyl and octyl. A C₁-C₆ straight chained or branched alkyl group isalso referred to as a “lower alkyl” group.

Moreover, the term “alkyl” (or “lower alkyl”) as used throughout thespecification, examples, and claims is intended to include both“unsubstituted alkyls” and “substituted alkyls”, the latter of whichrefers to alkyl moieties having substituents replacing a hydrogen on oneor more carbons of the hydrocarbon backbone. Such substituents, if nototherwise specified, can include, for example, a halogen, a hydroxyl, acarbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl),a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate),an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, anamino, an amido, an amidine, an imine, a cyano, a nitro, an azido, asulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, asulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic orheteroaromatic moiety. It will be understood by those skilled in the artthat the moieties substituted on the hydrocarbon chain can themselves besubstituted, if appropriate. For instance, the substituents of asubstituted alkyl may include substituted and unsubstituted forms ofamino, azido, imino, amido, phosphoryl (including phosphonate andphosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl andsulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls(including ketones, aldehydes, carboxylates, and esters), —CF₃, —CN andthe like. Exemplary substituted alkyls are described below. Cycloalkylscan be further substituted with alkyls, alkenyls, alkoxys, alkylthios,aminoalkyls, carbonyl-substituted alkyls, —CF₃, —CN, and the like.

The term “C_(x-y)” when used in conjunction with a chemical moiety, suchas, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant toinclude groups that contain from x to y carbons in the chain. Forexample, the term “C_(x-y)alkyl” refers to substituted or unsubstitutedsaturated hydrocarbon groups, including straight-chain alkyl andbranched-chain alkyl groups that contain from x to y carbons in thechain, including haloalkyl groups such as trifluoromethyl and2,2,2-tirfluoroethyl, etc. C₀ alkyl indicates a hydrogen where the groupis in a terminal position, a bond if internal. The terms“C_(2-y)alkenyl” and “C_(2-y)alkynyl” refer to substituted orunsubstituted unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but that contain atleast one double or triple bond respectively.

The term “alkylamino”, as used herein, refers to an amino groupsubstituted with at least one alkyl group.

The term “alkylthio”, as used herein, refers to a thiol groupsubstituted with an alkyl group and may be represented by the generalformula alkylS—.

The term “alkynyl”, as used herein, refers to an aliphatic groupcontaining at least one triple bond and is intended to include both“unsubstituted alkynyls” and “substituted alkynyls”, the latter of whichrefers to alkynyl moieties having substituents replacing a hydrogen onone or more carbons of the alkynyl group. Such substituents may occur onone or more carbons that are included or not included in one or moretriple bonds. Moreover, such substituents include all those contemplatedfor alkyl groups, as discussed above, except where stability isprohibitive. For example, substitution of alkynyl groups by one or morealkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups iscontemplated.

The term “amide”, as used herein, refers to a group

wherein each R¹⁰ independently represent a hydrogen or hydrocarbylgroup, or two R¹⁰ are taken together with the N atom to which they areattached complete a heterocycle having from 4 to 8 atoms in the ringstructure.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines and salts thereof, e.g., a moietythat can be represented by

wherein each R¹⁰ independently represents a hydrogen or a hydrocarbylgroup, or two R¹⁰ are taken together with the N atom to which they areattached complete a heterocycle having from 4 to 8 atoms in the ringstructure.

The term “aminoalkyl”, as used herein, refers to an alkyl groupsubstituted with an amino group.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group.

The term “aryl” as used herein include substituted or unsubstitutedsingle-ring aromatic groups in which each atom of the ring is carbon.Preferably the ring is a 5- to 7-membered ring, more preferably a6-membered ring. The term “aryl” also includes polycyclic ring systemshaving two or more cyclic rings in which two or more carbons are commonto two adjoining rings wherein at least one of the rings is aromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groupsinclude benzene, naphthalene, phenanthrene, phenol, aniline, and thelike.

The term “carbamate” is art-recognized and refers to a group

wherein R⁹ and R¹⁰ independently represent hydrogen or a hydrocarbylgroup, such as an alkyl group, or R⁹ and R¹⁰ taken together with theintervening atom(s) complete a heterocycle having from 4 to 8 atoms inthe ring structure.

The terms “carbocycle”, and “carbocyclic”, as used herein, refers to asaturated or unsaturated ring in which each atom of the ring is carbon.The term carbocycle includes both aromatic carbocycles and non-aromaticcarbocycles. Non-aromatic carbocycles include both cycloalkane rings, inwhich all carbon atoms are saturated, and cycloalkene rings, whichcontain at least one double bond. “Carbocycle” includes 5-7 memberedmonocyclic and 8-12 membered bicyclic rings. Each ring of a bicycliccarbocycle may be selected from saturated, unsaturated and aromaticrings. Carbocycle includes bicyclic molecules in which one, two or threeor more atoms are shared between the two rings. The term “fusedcarbocycle” refers to a bicyclic carbocycle in which each of the ringsshares two adjacent atoms with the other ring. Each ring of a fusedcarbocycle may be selected from saturated, unsaturated and aromaticrings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, maybe fused to a saturated or unsaturated ring, e.g., cyclohexane,cyclopentane, or cyclohexene. Any combination of saturated, unsaturatedand aromatic bicyclic rings, as valence permits, is included in thedefinition of carbocyclic. Exemplary “carbocycles” include cyclopentane,cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene,1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene andadamantane. Exemplary fused carbocycles include decalin, naphthalene,1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane,4,5,6,7-tetrahydro-1H-indene and bicyclo[4.1.0]hept-3-ene. “Carbocycles”may be substituted at any one or more positions capable of bearing ahydrogen atom.

A “cycloalkyl” group is a cyclic hydrocarbon which is completelysaturated. “Cycloalkyl” includes monocyclic and bicyclic rings.Typically, a monocyclic cycloalkyl group has from 3 to about 10 carbonatoms, more typically 3 to 8 carbon atoms unless otherwise defined. Thesecond ring of a bicyclic cycloalkyl may be selected from saturated,unsaturated and aromatic rings. Cycloalkyl includes bicyclic moleculesin which one, two or three or more atoms are shared between the tworings. The term “fused cycloalkyl” refers to a bicyclic cycloalkyl inwhich each of the rings shares two adjacent atoms with the other ring.The second ring of a fused bicyclic cycloalkyl may be selected fromsaturated, unsaturated and aromatic rings. A “cycloalkenyl” group is acyclic hydrocarbon containing one or more double bonds.

The term “carbocyclylalkyl”, as used herein, refers to an alkyl groupsubstituted with a carbocycle group.

The term “carbonate” is art-recognized and refers to a group —OCO₂—R¹⁰,wherein R¹⁰ represents a hydrocarbyl group.

The term “carboxy”, as used herein, refers to a group represented by theformula —CO₂H.

The term “ester”, as used herein, refers to a group —C(O)OR¹⁰ whereinR¹⁰ represents a hydrocarbyl group.

The term “ether”, as used herein, refers to a hydrocarbyl group linkedthrough an oxygen to another hydrocarbyl group. Accordingly, an ethersubstituent of a hydrocarbyl group may be hydrocarbyl-O—. Ethers may beeither symmetrical or unsymmetrical. Examples of ethers include, but arenot limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethersinclude “alkoxyalkyl” groups, which may be represented by the generalformula alkyl-O-alkyl.

The terms “halo” and “halogen” as used herein means halogen and includeschloro, fluoro, bromo, and iodo.

The terms “hetaralkyl” and “heteroaralkyl”, as used herein, refers to analkyl group substituted with a hetaryl group.

The term “heteroalkyl”, as used herein, refers to a saturated orunsaturated chain of carbon atoms and at least one heteroatom, whereinno two heteroatoms are adjacent.

The terms “heteroaryl” and “hetaryl” include substituted orunsubstituted aromatic single ring structures, preferably 5- to7-membered rings, more preferably 5- to 6-membered rings, whose ringstructures include at least one heteroatom, preferably one to fourheteroatoms, more preferably one or two heteroatoms. The terms“heteroaryl” and “hetaryl” also include polycyclic ring systems havingtwo or more cyclic rings in which two or more carbons are common to twoadjoining rings wherein at least one of the rings is heteroaromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroarylgroups include, for example, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, andpyrimidine, and the like.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, andsulfur.

The terms “heterocyclyl”, “heterocycle”, and “heterocyclic” refer tosubstituted or unsubstituted non-aromatic ring structures, preferably 3-to 10-membered rings, more preferably 3- to 7-membered rings, whose ringstructures include at least one heteroatom, preferably one to fourheteroatoms, more preferably one or two heteroatoms. The terms“heterocyclyl” and “heterocyclic” also include polycyclic ring systemshaving two or more cyclic rings in which two or more carbons are commonto two adjoining rings wherein at least one of the rings isheterocyclic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.Heterocyclyl groups include, for example, piperidine, piperazine,pyrrolidine, morpholine, lactones, lactams, and the like.

The term “heterocyclylalkyl”, as used herein, refers to an alkyl groupsubstituted with a heterocycle group.

The term “hydrocarbyl”, as used herein, refers to a group that is bondedthrough a carbon atom that does not have a ═O or ═S substituent, andtypically has at least one carbon-hydrogen bond and a primarily carbonbackbone, but may optionally include heteroatoms. Thus, groups likemethyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered to behydrocarbyl for the purposes of this application, but substituents suchas acetyl (which has a ═O substituent on the linking carbon) and ethoxy(which is linked through oxygen, not carbon) are not. Hydrocarbyl groupsinclude, but are not limited to aryl, heteroaryl, carbocycle,heterocyclyl, alkyl, alkenyl, alkynyl, and combinations thereof.

The term “hydroxyalkyl”, as used herein, refers to an alkyl groupsubstituted with a hydroxy group.

The term “lower” when used in conjunction with a chemical moiety, suchas, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant toinclude groups where there are ten or fewer non-hydrogen atoms in thesubstituent, preferably six or fewer. A “lower alkyl”, for example,refers to an alkyl group that contains ten or fewer carbon atoms,preferably six or fewer. In certain embodiments, acyl, acyloxy, alkyl,alkenyl, alkynyl, or alkoxy substituents defined herein are respectivelylower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, orlower alkoxy, whether they appear alone or in combination with othersubstituents, such as in the recitations hydroxyalkyl and aralkyl (inwhich case, for example, the atoms within the aryl group are not countedwhen counting the carbon atoms in the alkyl substituent).

The term “natural amino acid” refers to one of the twenty natural aminoacids: alanine, arginine, asparagine, aspartic acid, cysteine,glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, or valine. As used herein, the term “natural aminoacid” encompasses all stereoisomers, such as the D- and theL-stereoisomers. The term “natural amino acid side chain” refers to oneof the side chains on the twenty natural amino acids, that is, thesubstituent on the α-carbon or, in the case of proline, the propylenemoiety linking the α-carbon with the amino group.

The terms “polycyclyl”, “polycycle”, and “polycyclic” refer to two ormore rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls,heteroaryls, and/or heterocyclyls) in which two or more atoms are commonto two adjoining rings, e.g., the rings are “fused rings”. Each of therings of the polycycle can be substituted or unsubstituted. In certainembodiments, each ring of the polycycle contains from 3 to 10 atoms inthe ring, preferably from 5 to 7.

The term “silyl” refers to a silicon moiety with three hydrocarbylmoieties attached thereto.

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons of the backbone. It will be understoodthat “substitution” or “substituted with” includes the implicit provisothat such substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and non-aromaticsubstituents of organic compounds. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this invention, the heteroatoms such as nitrogen mayhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. Substituents can include any substituents described herein,for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, analkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as athioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, aphosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine,an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, asulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, aheterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. Itwill be understood by those skilled in the art that substituents canthemselves be substituted, if appropriate. Unless specifically stated as“unsubstituted,” references to chemical moieties herein are understoodto include substituted variants. For example, reference to an “aryl”group or moiety implicitly includes both substituted and unsubstitutedvariants.

The term “sulfate” is art-recognized and refers to the group —OSO₃H, ora pharmaceutically acceptable salt thereof.

The term “sulfonamide” is art-recognized and refers to the grouprepresented by the general formulae

wherein R⁹ and R¹⁰ independently represents hydrogen or hydrocarbyl,such as alkyl, or R⁹ and R¹⁰ taken together with the intervening atom(s)complete a heterocycle having from 4 to 8 atoms in the ring structure.

The term “sulfoxide” is art-recognized and refers to the group—S(O)—R¹⁰, wherein R¹⁰ represents a hydrocarbyl.

The term “sulfonate” is art-recognized and refers to the group SO₃H, ora pharmaceutically acceptable salt thereof.

The term “sulfone” is art-recognized and refers to the group —S(O)₂—R¹⁰,wherein R¹⁰ represents a hydrocarbyl.

The term “thioalkyl”, as used herein, refers to an alkyl groupsubstituted with a thiol group.

The term “thioester”, as used herein, refers to a group —C(O)SR¹⁰ or—SC(O)R¹⁰ wherein R¹⁰ represents a hydrocarbyl.

The term “thioether”, as used herein, is equivalent to an ether, whereinthe oxygen is replaced with a sulfur.

The term “urea” is art-recognized and may be represented by the generalformula

wherein R⁹ and R¹⁰ independently represent hydrogen or a hydrocarbyl,such as alkyl, or either occurrence of R⁹ taken together with R¹⁰ andthe intervening atom(s) complete a heterocycle having from 4 to 8 atomsin the ring structure.

“Protecting group” refers to a group of atoms that, when attached to areactive functional group in a molecule, mask, reduce or prevent thereactivity of the functional group. Typically, a protecting group may beselectively removed as desired during the course of a synthesis.Examples of protecting groups can be found in Greene and Wuts,Protective Groups in Organic Chemistry, 3^(rd) Ed., 1999, John Wiley &Sons, NY and Harrison et al., Compendium of Synthetic Organic Methods,Vols. 1-8, 1971-1996, John Wiley & Sons, NY. Representative nitrogenprotecting groups include, but are not limited to, formyl, acetyl,trifluoroacetyl, benzyl, benzyloxycarbonyl (“CBZ”), tert-butoxycarbonyl(“Boc”), trimethylsilyl (“TMS”), 2-trimethylsilyl-ethanesulfonyl(“TES”), trityl and substituted trityl groups, allyloxycarbonyl,9-fluorenylmethyloxycarbonyl (“FMOC”), nitro-veratryloxycarbonyl(“NVOC”) and the like. Representative hydroxylprotecting groups include,but are not limited to, those where the hydroxyl group is eitheracylated (esterified) or alkylated such as benzyl and trityl ethers, aswell as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers(e.g., TMS or TIPS groups), glycol ethers, such as ethylene glycol andpropylene glycol derivatives and allyl ethers.

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample. For example, a compound that preventsepilepsy may reduce the frequency of seizures and/or reduce the severityof seizures.

The term “treating” includes prophylactic and/or therapeutic treatments.The term “prophylactic or therapeutic” treatment is art-recognized andincludes administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic (i.e., it protects thehost against developing the unwanted condition), whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic, (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

The phrases “conjoint administration” and “administered conjointly”refer to any form of administration of two or more different therapeuticcompounds such that the second compound is administered while thepreviously administered therapeutic compound is still effective in thebody (e.g., the two compounds are simultaneously effective in thepatient, which may include synergistic effects of the two compounds).For example, the different therapeutic compounds can be administeredeither in the same formulation or in a separate formulation, eitherconcomitantly or sequentially. In certain embodiments, the differenttherapeutic compounds can be administered within one hour, 12 hours, 24hours, 36 hours, 48 hours, 72 hours, or a week of one another. Thus, anindividual who receives such treatment can benefit from a combinedeffect of different therapeutic compounds.

The term “prodrug” is intended to encompass compounds which, underphysiologic conditions, are converted into the therapeutically activeagents of the present invention (e.g., a compound selected from TableI). A common method for making a prodrug is to include one or moreselected moieties which are hydrolyzed under physiologic conditions toreveal the desired molecule. In other embodiments, the prodrug isconverted by an enzymatic activity of the host animal. For example,esters or carbonates (e.g., esters or carbonates of alcohols orcarboxylic acids) are preferred prodrugs of the present invention. Incertain embodiments, some or all of the compounds selected from Table Iin a formulation represented above can be replaced with thecorresponding suitable prodrug, e.g., wherein a hydroxyl in the parentcompound is presented as an ester or a carbonate or carboxylic acidpresent in the parent compound is presented as an ester.

Use of Deoxynucleotide Prodrugs

In some embodiments, the present invention provides a method of treatinga patient suffering from MDS, comprising administering to the patient atherapeutically effective amount of a compound of Formula (I) or (Ia).In some embodiments, the MDS is selected from DGUOK deficiency, TK2deficiency, MNGIE, POLG deficiency, Alpers-Huttenlocher syndrome, SANDOsyndrome, MIRAS, MPV17-related hepatocerebral myopathy, or RRM2B-relatedmyopathy. In some embodiments, the MDS is an RRM2B-related myopathy. Insome embodiments, the MDS is linked to a mutation in TK2, DGUOK, POLG,MPV17, RRM2B, SUCLA2, SUCLG1, TYMP, C10orf2, or SAMHD1. In someembodiments, the MDS has unknown pathophysiology.

In some embodiments, the dAMP and dGMP prodrugs of the presentinvention, i.e., the compounds of Formula (I) or (Ia) wherein NT isadenine or guanine, may be used to treat DGUOK deficiency.

In some embodiments, the dAMP and dGMP prodrugs of the presentinvention, i.e., the compounds of Formula (I) or (Ia) wherein NT isadenine or guanine or an adenine or guanine prodrug moiety, may be usedto treat DGUOK deficiency.

In other embodiments, the dCTP and dTTP prodrugs of the presentinvention, i.e., the compounds of Formula (I) or (Ia) wherein NT iscytosine or thymine, may be used to treat TK2 deficiency.

In other embodiments, the dCTP and dTTP prodrugs of the presentinvention, i.e., the compounds of Formula (I) or (Ia) wherein NT iscytosine or thymine or a cytosine or thymine prodrug moiety, may be usedto treat TK2 deficiency.

In certain embodiments, the dCTP prodrugs of the present invention,i.e., the compounds of Formula (I) or (Ia) wherein NT is cytosine, maybe used to treat MNGIE.

In certain embodiments, the dCTP prodrugs of the present invention,i.e., the compounds of Formula (I) or (Ia) wherein NT is cytosine or acytosine prodrug moiety, may be used to treat MNGIE.

In some embodiments, the dAMP, dGMP, dCTP, and dTTP prodrugs of thepresent invention, i.e., the compounds of Formula (I) or (Ia) wherein NTis adenine, guanine, cytosine, or thymine, may be used to treat POLGdeficiency. In certain such embodiments, NT is adenine or guanine.

In some embodiments, the dAMP, dGMP, dCTP, and dTTP prodrugs of thepresent invention, i.e., the compounds of Formula (I) or (Ia) wherein NTis adenine, guanine, cytosine, or thymine or an adenine, guanine,cytosine, or thymine prodrug moiety, may be used to treat POLGdeficiency. In certain such embodiments, NT is adenine or guanine or anadenine or guanine prodrug moiety.

In some embodiments, the dAMP and dGMP prodrugs of the presentinvention, i.e., the compounds of Formula (I) or (Ia) wherein NT isadenine or guanine or an adenine or guanine prodrug moiety, may be usedto treat MPV17. In certain such embodiments, NT is adenine or guanine.

In some embodiments, the dAMP, dGMP, dCTP, and dTTP prodrugs of thepresent invention, i.e., the compounds of Formula (I) or (Ia) wherein NTis adenine, guanine, cytosine, or thymine or an adenine, guanine,cytosine, or thymine prodrug moiety, may be used to treat amitochondrial DNA depletion syndrome that is linked to a mutation inSAMDH1. In certain such embodiments, NT is adenine, guanine, thymine orcytosine.

In some embodiments, the dAMP, dGMP, dCTP, and dTTP prodrugs of thepresent invention, i.e., the compounds of Formula (I) or (Ia) wherein NTis adenine, guanine, cytosine, or thymine or an adenine, guanine,cytosine, or thymine prodrug moiety, may be used to treat amitochondrial DNA depletion syndrome that is linked to a mutation inRR2MB. In certain such embodiments, NT is adenine, guanine, thymine orcytosine.

Pharmaceutical Compositions

The compositions and methods of the present invention may be utilized totreat an individual in need thereof. In certain embodiments, theindividual is a mammal such as a human, or a non-human mammal. Whenadministered to an animal, such as a human, the composition or thecompound is preferably administered as a pharmaceutical compositioncomprising, for example, a compound of the invention and apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers are well known in the art and include, for example, aqueoussolutions such as water or physiologically buffered saline or othersolvents or vehicles such as glycols, glycerol, oils such as olive oil,or injectable organic esters. In a preferred embodiment, when suchpharmaceutical compositions are for human administration, particularlyfor invasive routes of administration (i.e., routes, such as injectionor implantation, that circumvent transport or diffusion through anepithelial barrier), the aqueous solution is pyrogen-free, orsubstantially pyrogen-free. The excipients can be chosen, for example,to effect delayed release of an agent or to selectively target one ormore cells, tissues or organs. The pharmaceutical composition can be indosage unit form such as tablet, capsule (including sprinkle capsule andgelatin capsule), granule, lyophile for reconstitution, powder,solution, syrup, suppository, injection or the like. The composition canalso be present in a transdermal delivery system, e.g., a skin patch.The composition can also be present in a solution suitable for topicaladministration, such as an eye drop.

A pharmaceutically acceptable carrier can contain physiologicallyacceptable agents that act, for example, to stabilize, increasesolubility or to increase the absorption of a compound such as acompound of the invention. Such physiologically acceptable agentsinclude, for example, carbohydrates, such as glucose, sucrose ordextrans, antioxidants, such as ascorbic acid or glutathione, chelatingagents, low molecular weight proteins or other stabilizers orexcipients. The choice of a pharmaceutically acceptable carrier,including a physiologically acceptable agent, depends, for example, onthe route of administration of the composition. The preparation orpharmaceutical composition can be a selfemulsifying drug delivery systemor a selfmicroemulsifying drug delivery system. The pharmaceuticalcomposition (preparation) also can be a liposome or other polymermatrix, which can have incorporated therein, for example, a compound ofthe invention. Liposomes, for example, which comprise phospholipids orother lipids, are nontoxic, physiologically acceptable and metabolizablecarriers that are relatively simple to make and administer.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

A pharmaceutical composition (preparation) can be administered to asubject by any of a number of routes of administration including, forexample, orally (for example, drenches as in aqueous or non-aqueoussolutions or suspensions, tablets, capsules (including sprinkle capsulesand gelatin capsules), boluses, powders, granules, pastes forapplication to the tongue); absorption through the oral mucosa (e.g.,sublingually); anally, rectally or vaginally (for example, as a pessary,cream or foam); parenterally (including intramuscularly, intravenously,subcutaneously or intrathecally as, for example, a sterile solution orsuspension); nasally; intraperitoneally; subcutaneously; transdermally(for example as a patch applied to the skin); and topically (forexample, as a cream, ointment or spray applied to the skin, or as an eyedrop). The compound may also be formulated for inhalation. In certainembodiments, a compound may be simply dissolved or suspended in sterilewater. Details of appropriate routes of administration and compositionssuitable for same can be found in, for example, U.S. Pat. Nos.6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and4,172,896, as well as in patents cited therein.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thehost being treated, the particular mode of administration. The amount ofactive ingredient that can be combined with a carrier material toproduce a single dosage form will generally be that amount of thecompound which produces a therapeutic effect. Generally, out of onehundred percent, this amount will range from about 1 percent to aboutninety-nine percent of active ingredient, preferably from about 5percent to about 70 percent, most preferably from about 10 percent toabout 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association an active compound, such as a compound ofthe invention, with the carrier and, optionally, one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association a compound of the present inventionwith liquid carriers, or finely divided solid carriers, or both, andthen, if necessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules (including sprinkle capsules and gelatin capsules),cachets, pills, tablets, lozenges (using a flavored basis, usuallysucrose and acacia or tragacanth), lyophile, powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia) and/or as mouth washes and the like, each containinga predetermined amount of a compound of the present invention as anactive ingredient. Compositions or compounds may also be administered asa bolus, electuary or paste.

To prepare solid dosage forms for oral administration (capsules(including sprinkle capsules and gelatin capsules), tablets, pills,dragees, powders, granules and the like), the active ingredient is mixedwith one or more pharmaceutically acceptable carriers, such as sodiumcitrate or dicalcium phosphate, and/or any of the following: (1) fillersor extenders, such as starches, lactose, sucrose, glucose, mannitol,and/or silicic acid; (2) binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate;(5) solution retarding agents, such as paraffin; (6) absorptionaccelerators, such as quaternary ammonium compounds; (7) wetting agents,such as, for example, cetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such atalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof; (10) complexing agents,such as, modified and unmodified cyclodextrins; and (11) coloringagents. In the case of capsules (including sprinkle capsules and gelatincapsules), tablets and pills, the pharmaceutical compositions may alsocomprise buffering agents. Solid compositions of a similar type may alsobe employed as fillers in soft and hard-filled gelatin capsules usingsuch excipients as lactose or milk sugars, as well as high molecularweight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions, such as dragees, capsules (including sprinkle capsules andgelatin capsules), pills and granules, may optionally be scored orprepared with coatings and shells, such as enteric coatings and othercoatings well known in the pharmaceutical-formulating art. They may alsobe formulated so as to provide slow or controlled release of the activeingredient therein using, for example, hydroxypropylmethyl cellulose invarying proportions to provide the desired release profile, otherpolymer matrices, liposomes and/or microspheres. They may be sterilizedby, for example, filtration through a bacteria-retaining filter, or byincorporating sterilizing agents in the form of sterile solidcompositions that can be dissolved in sterile water, or some othersterile injectable medium immediately before use. These compositions mayalso optionally contain opacifying agents and may be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain portion of the gastrointestinal tract, optionally, in a delayedmanner. Examples of embedding compositions that can be used includepolymeric substances and waxes. The active ingredient can also be inmicroencapsulated form, if appropriate, with one or more of theabove-described excipients.

Liquid dosage forms useful for oral administration includepharmaceutically acceptable emulsions, lyophiles for reconstitution,microemulsions, solutions, suspensions, syrups and elixirs. In additionto the active ingredient, the liquid dosage forms may contain inertdiluents commonly used in the art, such as, for example, water or othersolvents, cyclodextrins and derivatives thereof, solubilizing agents andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions for rectal, vaginal, orurethral administration may be presented as a suppository, which may beprepared by mixing one or more active compounds with one or moresuitable nonirritating excipients or carriers comprising, for example,cocoa butter, polyethylene glycol, a suppository wax or a salicylate,and which is solid at room temperature, but liquid at body temperatureand, therefore, will melt in the rectum or vaginal cavity and releasethe active compound.

Formulations of the pharmaceutical compositions for administration tothe mouth may be presented as a mouthwash, or an oral spray, or an oralointment.

Alternatively or additionally, compositions can be formulated fordelivery via a catheter, stent, wire, or other intraluminal device.Delivery via such devices may be especially useful for delivery to thebladder, urethra, ureter, rectum, or intestine.

Formulations which are suitable for vaginal administration also includepessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches and inhalants. The active compound may be mixed under sterileconditions with a pharmaceutically acceptable carrier, and with anypreservatives, buffers, or propellants that may be required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound, excipients, such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays can contain, in addition to an active compound,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates and polyamide powder, or mixtures of these substances.Sprays can additionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the active compound in theproper medium. Absorption enhancers can also be used to increase theflux of the compound across the skin. The rate of such flux can becontrolled by either providing a rate controlling membrane or dispersingthe compound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.Exemplary ophthalmic formulations are described in U.S. Publication Nos.2005/0080056, 2005/0059744, 2005/0031697 and 2005/004074 and U.S. Pat.No. 6,583,124, the contents of which are incorporated herein byreference. If desired, liquid ophthalmic formulations have propertiessimilar to that of lacrimal fluids, aqueous humor or vitreous humor orare compatible with such fluids. A preferred route of administration islocal administration (e.g., topical administration, such as eye drops,or administration via an implant).

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.Pharmaceutical compositions suitable for parenteral administrationcomprise one or more active compounds in combination with one or morepharmaceutically acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents that delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsulated matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissue.

For use in the methods of this invention, active compounds can be givenper se or as a pharmaceutical composition containing, for example, 0.1to 99.5% (more preferably, 0.5 to 90%) of active ingredient incombination with a pharmaceutically acceptable carrier.

Methods of introduction may also be provided by rechargeable orbiodegradable devices. Various slow release polymeric devices have beendeveloped and tested in vivo in recent years for the controlled deliveryof drugs, including proteinaceous biopharmaceuticals. A variety ofbiocompatible polymers (including hydrogels), including bothbiodegradable and non-degradable polymers, can be used to form animplant for the sustained release of a compound at a particular targetsite.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions may be varied so as to obtain an amount of the activeingredient that is effective to achieve the desired therapeutic responsefor a particular patient, composition, and mode of administration,without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound or combination ofcompounds employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound(s) being employed, the duration of the treatment,other drugs, compounds and/or materials used in combination with theparticular compound(s) employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the therapeutically effective amount of thepharmaceutical composition required. For example, the physician orveterinarian could start doses of the pharmaceutical composition orcompound at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved. By “therapeutically effective amount” ismeant the concentration of a compound that is sufficient to elicit thedesired therapeutic effect. It is generally understood that theeffective amount of the compound will vary according to the weight, sex,age, and medical history of the subject. Other factors which influencethe effective amount may include, but are not limited to, the severityof the patient's condition, the disorder being treated, the stability ofthe compound, and, if desired, another type of therapeutic agent beingadministered with the compound of the invention. A larger total dose canbe delivered by multiple administrations of the agent. Methods todetermine efficacy and dosage are known to those skilled in the art(Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13ed., 1814-1882, herein incorporated by reference).

In general, a suitable daily dose of an active compound used in thecompositions and methods of the invention will be that amount of thecompound that is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above.

If desired, the effective daily dose of the active compound may beadministered as one, two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms. In certain embodiments of the presentinvention, the active compound may be administered two or three timesdaily. In preferred embodiments, the active compound will beadministered once daily.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

In certain embodiments, compounds of the invention may be used alone orconjointly administered with another type of therapeutic agent.

This invention includes the use of pharmaceutically acceptable salts ofcompounds of the invention in the compositions and methods of thepresent invention. In certain embodiments, contemplated salts of theinvention include, but are not limited to, alkyl, dialkyl, trialkyl ortetra-alkyl ammonium salts. In certain embodiments, contemplated saltsof the invention include, but are not limited to, L-arginine,benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol,diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine,ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, lithium,L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine,potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine,tromethamine, and zinc salts. In certain embodiments, contemplated saltsof the invention include, but are not limited to, Na, Ca, K, Mg, Zn orother metal salts.

The pharmaceutically acceptable acid addition salts can also exist asvarious solvates, such as with water, methanol, ethanol,dimethylformamide, and the like. Mixtures of such solvates can also beprepared. The source of such solvate can be from the solvent ofcrystallization, inherent in the solvent of preparation orcrystallization, or adventitious to such solvent.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1)water-soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and (3)metal-chelating agents, such as citric acid, ethylenediamine tetraaceticacid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

In certain embodiments, the invention relates to a method for conductinga pharmaceutical business, by manufacturing a formulation of a compoundof the invention, or a kit as described herein, and marketing tohealthcare providers the benefits of using the formulation or kit fortreating or preventing any of the diseases or conditions as describedherein.

Exemplification

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

EXAMPLES Example 1 Synthetic Protocols (Method A)

General Procedure for the Preparation of Compound 12 isopropyl((((2R,3S,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate

To a solution of compound 1001 (25.0 g, 118.5 mmol, 1.0 eq) indichloromethane (250 mL) at −78° C. was added a solution of4-nitrophenol (16.5 g, 118.5 mmol, 1.0 eq) in dichloromethane (250 mL)and TEA (18 mL, 130.3 mmol, 1.1 eq). The reaction mixture was warmed toroom temperature, stirred for 1 h, and cooled to 0° C. A solution ofcompound 1003 (19.9 g, 118.5 mmol, 1.0 eq) and triethylamine (34.5 mL,248.9 mmol, 2.1 eq) in dichloromethane (250 mL) was added. The mixturewas warmed to room temperature, stirred for 2 h, and quenched with water(500 mL). The organic layer was separated, dried over sodium sulfate,filtered, and concentrated under reduced pressure. The crude product waspurified by flash column chromatography on silica gel (Et₂O/EtOAc=2/1)to give compound 1004 (25.0 g, 52%) as colorless oil. LC-MS: 409.2[M+H]⁺, expected 409.11, ¹H NMR (400 MHz, CDCl₃) (δ, ppm) 8.18 (d, J=8.7Hz, 2H), 7.34 (ddd, J=15.9, 12.9, 5.1 Hz, 4H), 7.27-7.08 (m, 3H), 4.98(m, 1H), 4.36-4.16 (m, 1H), 1.35 (d, J=7.0 Hz, 3H), 1.24-1.15 (m, 6H).

To a solution of compound 1005 (2.6 g, 9.8 mmol, 1.0 eq) in THF (7.5 mL)and NMP (30 mL) at 0° C. was added 1.0 M t-BuMgCl (14.8 mL, 14.7 mmol,1.5 eq). The mixture was stirred at 0° C. for 0.5 h and a solution ofcompound 1004 (3.0 g, 7.35 mmol, 0.75 eq) in THF (10 mL) was added. Themixture was warmed to room temperature and stirred overnight. Asaturated aqueous solution of NH₄Cl (30 mL) was added and the organicphase was extracted with ethyl acetate (2+50 mL). The combined organiclayer was dried with sodium sulfate, filtered, and concentrated. Thecrude product was purified by column chromatography on silica gel twice(DCM−DCM/MeOH=15/1) to afford compound 12 (600 mg, 17%, >95% purity) asa white solid.

The Following Compounds were Prepared According to the General ProcedureDescribed in Method A via Displacement of the 4-nitrophenol LeavingGroup with the Appropriate deoxynucleoside Base.

Observed Comp. MW ¹H NMR No. IUPAC Name (Expected) (400 MHz) 12isopropyl ((((2R,3S,5R)-5-(2- 537.2 CD₃OD (δ, ppm) 7.97 (s, 1H), (G)amino-6-oxo-1,6-dihydro-9H- (537.3) 7.35-7.25 (m, 2H), 7.17 (td, J =15.9, purin-9-yl)-3- 7.5 Hz, 3H), 6.28 (dd, J = 13.6,hydroxytetrahydrofuran-2- 6.5 Hz, 1H), 4.59 (m,yl)methoxy)(phenoxy)phosphoryl)- 1H), 4.37 (m, 2H), 4.28 (m, L-alaninate1H), 4.16 (m, 1H), 3.86 (m, 1H), 2.66 (m, 1H), 2.40 (m, 1H), 1.28 (t, J= 6.4 Hz, 3H), 1.23-1.14 (m, 6H) 18 isopropyl ((((2R,3S,5R)-5-(2- 555.2CD₃OD (δ, ppm) 7.92 (m, 1H), (G) amino-6-oxo-1,6-dihydro-9H- (555.2)7.30-7.15 (m, 2H), 7.05 (m, purin-9-yl)-3- 2H), 6.28 (m, 1H), 4.59 (m,hydroxytetrahydrofuran-2- 1H), 4.37 (m, 2H), 4.28 (m, yl)methoxy)(4-1H), 4.16 (m, 1H), 3.84 (m, fluorophenoxy)phosphoryl)-L- 1H), 2.70 (m,1H), 2.38 (m, alaninate 1H), 1.24 (t, J = 6.4 Hz, 3H), 1.23-1.14 (m, 6H)15 isopropyl ((((2R,3S,5R)-5-(2- 587.2 CD₃OD (δ, ppm) 8.10 (m, 1H), (G)amino-6-oxo-1,6-dihydro-9H- (587.3) 7.88 (m, 1H), 7.68 (m, 1H),purin-9-yl)-3- 7.50 (m, 3H), 7.38 (m, 2H), hydroxytetrahydrofuran-2-6.24 (m, 1H), 4.55 (m, 1H), yl)methoxy)(naphthalen-1- 4.40 (m, 2H), 4.36(m, 1H), yloxy)phosphoryl)-L-alaninate 4.18 (m, 1H), 3.98 (m, 1H), 2.45(m, 1H), 2.30 (m, 1H), 1.28 (m, 3H), 1.20-1.14 (m, 6H) 21 isopropyl((((2R,3S,5R)-3- 512.2 CDCl₃ (δ, ppm) 8.85 (brs, 1H), (T)hydroxy-5-(5-methyl-2,4-dioxo- (512.3) 7.39-7.28 (m, 3H),3,4-dihydropyrimidin-1(2H)- 7.24-7.11 (m, 3H), 6.25 (m, 1H),yl)tetrahydrofuran-2- 5.08-4.90 (m, 1H), 4.48 (m, 1H),yl)methoxy)(phenoxy)phosphoryl)- 4.32 (m, 1H), 4.10-3.90 (m, L-alaninate3H), 3.80-3.60 (m, 1H), 2.42-2.29 (m, 1H), 2.12 (m, 1H), 1.89 (d, J =3.6 Hz, 3H), 1.77 (m, 1H), 1.35 (m, 3H), 1.22 (m, 6H) 27 isopropyl ((4-530.2 CDCl₃ (δ, ppm) 9.49 (s, 1H), (T) fluorophenoxy)(((2R,3S,5R)-3-(530.3) 7.35 (s, 1H), 7.16 (m, 2H), hydroxy-5-(5-methyl-2,4-dioxo- 6.99(m, 2H), 6.27 (m, 1H), 3,4-dihydropyrimidin-1(2H)- 5.05-4.84 (m, 1H),4.47 (m, 1H), yl)tetrahydrofuran-2- 4.31 (m, 2H), 4.21 (m, 1H),yl)methoxy)phosphoryl)-L- 4.08 (m, 1H), 4.00-3.87 (m, alaninate 1H),2.36 (s, 1H), 2.15 (m, 2H), 1.85 (d, J = 19.8 Hz, 3H), 1.34 (t, J = 7.6Hz, 3H), 1.20 (m, 6H) 24 isopropyl ((((2R,3S,5R)-3- 562.2 CDCl₃ (δ, ppm)8.85 (m, 1H), (T) hydroxy-5-(5-methyl-2,4-dioxo- (562.2) 8.05 (m, 1H),7.83 (m, 1H), 3,4-dihydropyrimidin-1(2H)- 7.65 (m, 1H), 7.57-7.44 (m,yl)tetrahydrofuran-2- 3H), 7.44-7.33 (m, 2H), yl)methoxy)(naphthalen-1-7.31 (m, 1H), 6.24 (m, 1H), yloxy)phosphoryl)-L-alaninate 5.01-4.87 (m,1H), 4.39 (m, 3H), 4.05 (m, 4H), 2.28 (m, 1H), 1.98 (m, 1H), 1.78 (d, J= 18.6 Hz, 3H), 1.32 (dd, J = 10.9, 6.8 Hz, 3H), 1.20 (d, J = 6.2 Hz,3H), 1.16 (dd, J = 9.4, 6.3 Hz, 3H) 30 isopropyl ((((2R,3S,5R)-5-(4-497.2 CD₃OD (δ, ppm) 7.82 (m, 1H), (C) amino-2-oxopyrimidin-1(2H)-yl)-(497.5) 7.38 (m, 2H), 7.22 (m, 3H), 3-hydroxytetrahydrofuran-2- 6.24 (m,1H), 5.84 (m, 1H), yl)methoxy)(phenoxy)phosphoryl)- 4.38 (m, 2H), 4.30(m, 2H), L-alaninate 4.14 (m, 1H), 3.90 (m, 1H), 2.32 (m, 1H), 1.98 (m,1H), 1.34 (m, 3H), 1.20 (m, 6H) 36 isopropyl ((((2R,3S,5R)-5-(4- 515.2CD₃OD (δ, ppm) 7.82 (m, 1H), (C) amino-2-oxopyrimidin-1(2H)-yl)- (515.4)7.28 (m, 2H), 7.08 (m, 3H), 3-hydroxytetrahydrofuran-2- 6.28 (m, 1H),5.94 (m, 1H), yl)methoxy)(4- 4.38 (m, 2H), 4.30 (m, 2H),fluorophenoxy)phosphoryl)-L- 4.10 (m, 1H), 3.90 (m, 1H), alaninate 2.36(m, 1H), 2.04 (m, 1H), 1.34 (m, 3H), 1.24 (m, 6H) 33 isopropyl((((2R,3S,5R)-5-(4- 547.2 CD₃OD (δ, ppm) 8.18 (m, 1H), (C)amino-2-oxopyrimidin-1(2H)-yl)- (547.3) 7.88 (m, 1H), 7.70 (m, 1H),3-hydroxytetrahydrofuran-2- 7.56 (m, 3H), 7.54 (m, 1H), 7.44 (m,yl)methoxy)(naphthalen-1- 1H), 6.20 (m, 1H), 5.76 (m,yloxy)phosphoryl)-L-alaninate 1H), 4.38 (m, 1H), 4.30 (m, 3H), 4.10 (m,1H), 3.98 (m, 1H), 2.18 (m, 1H), 1.64 (m, 1H), 1.34 (m, 3H), 1.22 (m,6H)

Example 1 Synthetic Protocols (Method B)

General Procedure for the Preparation of Compound 1017 isopropyl((((2R,3S,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3-hydroxytetrahydrofuran-2-yl)methoxy)(naphthalen-1-yloxy)phosphoryl)-L-alaninate

To a solution of compound 1005 (3.0 g, 11.2 mmol, 1.0 eq) in MeOH (200mL) at −20° C. was added excess CH₂N₂ etherate and stirred for 4 h. Thereaction was monitored by LCMS. The resulting mixture was concentrated,triturated with MeOH, and filtered. The filtrate was concentrated toafford crude compound 1015 (2.5 g, 79%) as white powder, which was usedfor next step without further purification. To a solution of compound1016 (2.0 g, 4.37 mmol, 1.0 eq) in THF (6 mL) and NMP (25 mL) at 0° C.was added 1.0 M t-BuMgCl (6.55 mL, 6.55 mmol, 1.5 eq). The mixture wasstirred at 0° C. for 0.5 h and a solution of compound 1015 (2.5 g, 8.9mmol, 2.04 eq) in THF (8 mL) was added. The mixture was warmed to roomtemperature and stirred for 16 h. A saturated aqueous solution of NH₄Cl(25 mL) was added and the organic phase was extracted with ethyl acetate(2×40 mL). The combined organic layer was dried over sodium sulfate,filtered, and concentrated. The crude product was dissolved in MeOH andpurified by prep-HPLC to afford compound 1017 (158 mg, 6%, >95% purity)as white solid. LCMS: m/z (ESI+) 601.3 [M+1]⁺, expected 601.2, ¹H NMR(400 MHz, DMSO-d6) δ8.09 (t, J=6.4 Hz, 1H), 7.95-7.87 (m, 2H), 7.71 (t,J=7.2 Hz, 1H), 7.56-7.43 (m, 2H), 7.40-7.35 (m, 2H), 6.44 (d, J=2.4 Hz,2H), 6.23-6.13 (m, 2H), 5.46 (t, J=4.8 Hz, 1H) 4.84-4.73 (m, 1H),4.42-4.39 (m, 1H), 4.34-4.28 (m, 1H), 4.24-4.18 (m, 1H), 4.12-4.04 (m,1H), 3.98 (s, 3H), 3.93-3.76 (m, 1H), 2.61-2.47 (m, 1H), 2.25-2.13 (m,1H), 1.19 (d, J=7.2 Hz, 3H), 1.09-1.03 (m, 6H).

Example 1 Synthetic Protocols (Method C)

General Procedure for the Preparation of Compound 1023 neopentyl((((2R,3S,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3-hydroxytetrahydrofuran-2-yl)methoxy)(naphthalen-1-yloxy)phosphoryl)-L-alaninate

To a mixture of compound 1018 (10 g, 52.8 mmol, 1.0 eq) and neopentylalcohol (5.58 g, 63.4 mmol, 1.2 eq) in DCM (100 mL) at 0° C. undernitrogen atmosphere was added DMAP (0.64 g, 5.28 mmol, 0.1 eq) and EDCl.HCl (15.2 g, 79.3 mmol, 1.5 eq). The reaction mixture was warmed to rtand stirred for 16 h. The reaction was monitored by TLC. The mixture wasextracted with ethyl acetate (3×100 mL). The organic phase was washedwith brine, dried over Na₂SO₄ and concentrated under reduced pressure.The residue was purified by flash chromatography on silica(Et₂O/EtOAc=30:1) to give compound 1019 (12.5 g, 91%) as colorless oil.

To a solution of compound 1019 (6.16 g, 23.8 mmol, 1.0 eq) in HCl/EtOAcsolution (2 M, 50 mL, 100 mmol) was stirred at rt for 1 h. The reactionwas monitored by ¹H NMR. The mixture was concentrated under reducedpressure to give compound 1020 (4.42 g, 95%) as white powder.

A mixture of compound 1020 (1.0 g, 5.1 mmol, 1.0 eq), compound 1021 (3.7g, 10.2 mmol, 2.0 eq) in DCM (10 mL) at 0° C. was added triethylamine(2.23 mL, 16.1 mmol, 3.15 eq). The reaction mixture was warmed to rt andstirred for 2 h. The reaction was monitored by TLC. Then the mixture wasextracted with EtOAc (3×20 mL). The organic phase was washed with brine,dried over sodium sulfate and concentrated under reduced pressure. Theresidue was purified by flash chromatography on silica(Et₂O/EtOAc=50:1-30:1-1:1) to give compound 1022 (650 mg, 26%) as awhite solid.

To a solution of compound 1022 (660 mg, 1.36 mmol, 1.0 eq) in THF (2.77mL) and NMP (8.32 mL) at 0° C. was added 1.0 M t-BuMgCl (4.09 mL, 4.08mmol, 3.0 eq). The mixture was stirred at 0° C. for 0.5 h and a solutionof compound 1005 (726 mg, 2.72 mmol, 2.0 eq) in THF (2.77 mL) was added.The mixture was warmed to rt and stirred for 16 h. The reaction wasmonitored by LCMS. A saturated aqueous solution of NH₄Cl (5 mL) wasadded and the organic phase was extracted with EtOAc (2×10 mL). Thecombined organic layers were dried over Na₂SO₄, filtered, andconcentrated. The crude product was purified by prep-HPLC to afford 1023(100 mg, 12%, >95% purity) as a white powder. LCMS: m/z (ESI+) 615.4[M+H]⁺, expected 615.2, ¹H NMR (400 MHz, DMSO-d6) δ 10.59 (d, J=2.8 Hz,1H), 8.10 (t, J=9.6 Hz, 1H), 7.94-7.89 (m, 1H), 7.78-7.56 (m, 2H),7.54-7.49 (m, 2H), 7.46-7.37 (m, 2H), 6.43 (d, J=4.0 Hz, 2H), 6.23-6.16(m, 1H), 6.13-6.09 (m, 1H), 5.40 (t, J=4.4 Hz, 1H), 4.38-4.22 (m, 1H),4.14-4.02 (m, 2H), 3.98-3.87 (m, 2H), 3.74-3.71 (m, 1H), 3.70-3.59 (m,1H), 2.48-2.33 (m, 1H), 2.21-2.11 (m, 1H), 1.25-1.22 (m, 3H), 0.82 (s,9H).

Example 1 Synthetic Protocols (Method D)

General Procedure for the Preparation of Compound 14 benzyl((((2R,3S,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3-hydroxytetrahydrofuran-2-yl)methoxy)(naphthalen-1-yloxy)phosphoryl)-L-alaninate

A solution of compound 1024 (10.0 g, 46.5 mmol, 1.0 eq), compound 1021(33.8 g, 93 mmol, 2.0 eq) and TEA (13.5 mL, 97.7 mmol, 2.1 eq) in DCM(120 mL) was stirred at 0° C. The mixture was warmed to rt and stirredfor 2 h. The reaction was monitored by LCMS. The resulting mixture wasquenched with water (250 mL). The organic layer was separated, driedover Na₂SO₄, filtered, and concentrated under reduced pressure. Thecrude product was purified by flash chromatography on silica(Et₂O/EtOAc=2/1) to give compound 1025 (14 g, 59%) as colorless oil.

To a solution of compound 1025 (5.0 g, 9.88 mmol, 1.0 eq) in THF (15 mL)and NMP (60 mL) at 0° C. was added 1.0 M t-BuMgCl (14.8 mL, 14.8 mmol,1.5 eq). The mixture was stirred at 0° C. for 0.5 h and a solution ofcompound 7a (1.98 g, 7.41 mmol, 0.75 eq) in THF (8 mL) was added. Themixture was warmed to rt and stirred for 16 h. A saturated aqueoussolution of NH₄Cl (60 mL) was added and the organic phase was extractedwith ethyl acetate (2×100 mL). The combined organic layer was dried overNa₂SO₄, filtered, and concentrated. The crude product was purified byprep-HPLC to afford compound 14 (180 mg, 3%, >95% purity) as whitesolid. LCMS: m/z (ESI+) 635.3 [M+H]⁺, expected 635.2, NMR (400 MHz,DMSO-d6) δ 10.57 (s, 1H), 8.09 (d, J=7.6 Hz, 1H), 7.92 (d, J=7.2 Hz,1H), 7.77 (s, 1H), 7.71 (d, J=7.6 Hz, 1H), 7.56-7.48 (m, 2H), 7.45-7.36(m, 2H), 7.28 (s, 5H), 6.43 (s, 2H), 6.29-6.22 (m, 1H), 6.12 (t, J=6.0Hz, 1H), 5.40 (d, J=4.0 Hz, 1H), 5.02 (dd, J=12.4, 12.4 Hz, 2H),4.38-4.32 (m, 1H), 4.26-4.20 (m, 1H), 4.12-4.05 (m, 1H), 4.01-3.93 (m,2H), 2.48-2.39 (m, 1H), 2.21-2.14 (m, 1H), 1.24-1.20 (m, 6H).

Example 2 Log P (pH 11.0) Assay and Caco-2 Permeability Assay

The Log P assay was performed according to a miniaturized1-octanol/buffer shake flask method followed by LC/MS/MS analysis. Testcompounds were prepared as 10 mM solutions dissolved in 100% DMSO. Testcompounds (10 mM in DMSO; 2 μL/well) and QC samples (10 mM in DMSO; 2μL/well) were transferred in duplicate from storage tubes to the 96-wellpolypropylene cluster tubes. Buffer was prepared as 80 mM phosphate, 80mM borate, and 80 mM acetate solution at pH 11.0 with 1% DMSO.Buffer-saturated 1-octanol (149 μL/well) and 1-octanol saturated buffer(149 μL/well) were added to each well. Each of the tubes was vigorouslymixed on their sides for 3 minutes and then shaken upright for 1 hour ata speed of 880 rpm at room temperature. The tubes were centrifuged at2500 rpm for 2 minutes. The buffer layer sample was diluted by a factorof 20 and the 1-octanol layer was diluted by a factor of 200 withinternal standard solution. Sample analysis was performed using a triplequadrupole mass spectrometer. Peak areas were corrected by dilutionfactors and by reference to an internal standard, and the ratio of thecorrected peak areas were used to calculate the results (Log P value).Data Analysis—The Log P value for each compound was calculated using thefollowing equation:

${{Log}\mspace{14mu} D_{{oct}/{buffer}}} = {\log\left( \frac{\left\lbrack {200 - {{fold}\mspace{14mu}{dilution}\mspace{14mu}{of}\mspace{14mu}{compound}}} \right\rbrack_{octanol} \times 200}{\left\lbrack {20 - {{fold}\mspace{11mu}{compound}}} \right\rbrack_{biffer} \times 20} \right)}$

The results are presented in Table 3.

The Caco-2 permeability assay was performed as follows.

Caco-2 cells purchased from ATCC were seeded onto polyethylene membranes(PET) in 96-well BD Insert plates at 1×10⁵ cells/cm², and refreshedmedium every 4-5 days until to the 21^(st) to 28^(th) day for confluentcell monolayer formation.

The transport buffer in the study was HBSS with 10 mM HEPES at pH7.40±0.05. Test compounds were tested at 2 μM in the presence or absenceof 30 μM novobiocin (BCRP inhibitor), verapamil (Pgp inhibitor), orGF120918 (BCRP/Pgp inhibitor) bi-directionally in duplicate. E3S controlwas tested at 5 μM in the presence or absence of efflux inhibitorsbi-directionally in duplicate, while fenoterol and propranolol controlswere tested at 2 μM in the absence of efflux inhibition in A to Bdirection in duplicate. Final DMSO concentration was adjusted to lessthan 1%. The plate was incubated for 120 minutes in a CO₂ incubator at37±1° C., with 5% CO₂ at saturated humidity without shaking. All sampleswere mixed with acetonitrile containing internal standard andcentrifuged at 4000 rpm for 20 min. Subsequently, 100 μL supernatantsolution was diluted with 100 μLt distilled water for LC/MS/MS analysis.Concentrations of test and control compounds in starting solution, donorsolution, and receiver solution were quantified by LC/MS/MS, using peakarea ratio of analyte/internal standard, and permeation of luciferyellow through the monolayer was measured to evaluate the cellularintegrity.

The apparent permeability coefficient P_(app) (cm/s) was calculatedusing the equation:P _(app)=(dC _(r) /dt)×V _(r)/(A×C ₀),where dC_(r)/dt is the slope of the cumulative concentration of compoundin the receiver chamber as a function of time (μM/s); V_(r) is thesolution volume in the receiver chamber (0.075 mL on the apical side,0.25 mL on the basolateral side); A is the surface area for thetransport, i.e. 0.0804 cm² for the area of the monolayer; and C₀ is theinitial concentration in the donor chamber (μM). Percent recovery wascalculated using the equation:% Recovery=100×[(V _(r) ×C _(r))+(V _(d) ×C _(d))]/(V _(d) ×C ₀),where V_(d) is the volume in the donor chambers (0.075 mL on the apicalside, 0.25 mL on the basolateral side); and C_(d) and C_(r) are thefinal concentrations of transport compound in donor and receiverchambers, respectively. The results are presented in Table 3.

TABLE 3 Log P and Caco-2 data for selected compounds Log P Caco-2P_(app) Compound (@ pH = 11) (A − B/B − A × 10⁻⁶)^(a) dTMP −1.9<0.03/<0.03 21 0.67 0.10/7.9  27 0.97 0.07/8.9  24 1.7 0.04/12.6 dCMP−2.1 <0.04/<0.04 30 1.2 0.14/0.57 36 1.5 0.24/0.66 33 2.1 0.19/1.39 dGMP−1.4  0.28/0.51^(b) 12 −0.03 0.17/0.64 18 0.24 0.16/0.57 15 1.10.10/1.76 1017  3.0 2.0/5.7 1023  1.7 0.06/0.49 14 3.4 0.04/0.31^(a)with efflux inhibitor, ^(b)recovery < 2%

Example 3 dNMP Prodrugs Rescue mtDNA Depletion in Patient-DerivedFibroblasts with DGUOK Deficiency

Patient-derived fibroblast cell line 10028 containing a DGUOK splicingvariant c.592-4_c.592-3delTT and a c.677A>G (p.H226R) resulting in asevere neonatal onset hepatocerebral presentation and mtDNA depletionwas used, as described in Buchaklian et. al., Molecular Genetics andMetabolism, 2012, 107, 92-94. Cells were cultured in 3.5-cm diameterplates containing αMEM with 10% FBS plus 20 mM L-glutamine. Onceconfluent, cells were supplemented with serum-starved αMEM plus 20 mML-glutamine. Compounds were dissolved in DMSO vehicle and added to mediacontaining cells to give a final concentration between 1 and 100 uM.Control cells were supplemented with vehicle only. Cells were incubatedwith compound or vehicle for 10 consecutive days in serum-starved media,which was exchanged daily with identical media containing freshlyprepared compound or vehicle. mtDNA copy number was assessed via qPCR asdescribed in Venegas et. al., Current Protocols in Human Genetics, 2011,Chapter 19, Unit 19.7. The results are presented in FIG. 1. Both of thedNMP prodrugs tested, compounds 15 and 1017, were found to increasemtDNA copy number relative to control in a dose-dependent manner.

Incorporation by Reference

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control.

Equivalents

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification and the claims below. The fullscope of the invention should be determined by reference to the claims,along with their full scope of equivalents, and the specification, alongwith such variations.

The invention claimed is:
 1. A compound having the structure of formula (I) or a pharmaceutically acceptable salt thereof:

wherein: R₁ is aryl or; R₂ and R₂′, each independently, are hydrogen, alkyl, or aralkyl; R₃ is alkyl or aralkyl; R₄ is hydrogen or alkyl; NT is a nucleobase.
 2. The compound of claim 1, wherein the nucleobase is selected from:

and further wherein R₅ is alkyl or aralkyl.
 3. The compound of claim 2, wherein R₅ is methyl, ethyl, isopropyl, or benzyl.
 4. The compound of claim 1, wherein R₁ is phenyl, naphthyl, or 4-fluorophenyl.
 5. The compound of claim 1, wherein R₂ is alkyl.
 6. The compound of claim 1, wherein R₂′ is H.
 7. The compound of claim 1, wherein R₃ is methyl, benzyl, neopentyl or isopropyl.
 8. The compound of formula (I) in claim 1, wherein the compound is:

or a pharmaceutically acceptable salt thereof.
 9. The compound of claim 1, having the structure of formula (I) or a pharmaceutically acceptable salt thereof:

wherein: R₁ is aryl; R₂ is hydrogen, or alkyl or aralkyl; R₃ is alkyl or aralkyl; R₄ is hydrogen or alkyl; and NT is 9-adeninyl, 9-guaninyl, 1-cytosinyl, or 1-thyminyl.
 10. A pharmaceutical composition comprising a compound of claim 1 in combination with a pharmaceutically acceptable carrier. 